A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.
A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.
One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.
Part 1: Injecting Zebrafish Embryos at the 1-cell stage.
Part 2: Making chimeric zebrafish embryos by transplantation.
Figure 1:Microscope set-up for making chimeric embryos. A: A dissecting microscope transplant rig consists of an oil-filled Hamilton syringe with a micrometer drive (a) connected to an oil-filled reservoir (b) and the transplant pipette (c) via a 3-way stopcock (d). The transplant pipette is mounted on a coarse micromanipulator (e) which is attached to a metal base plate (not shown) via a magnetic foot (f). Transplants are performed on the stage of a stereomicroscope (g) equipped with bottom lighting for optimal optics. B: a compound microscope transplant rig consists of a similar oil-filled Hamilton syringe and micrometer drive (a,b) but in this case the transplant pipette is mounted on a pipette holder (c) whose X, Y and Z movements can be controlled either by a coarse (d) or fine (e) micromanipulator. In this example, the micromanipulator is mounted on the body of a fixed-stage microscope, however it is also possible to mount it on the stage of a regular microscope. The embryos, which are immobilized in methylcellulose on a depression slide, are visualized using a 10x objective (f). C: Transplant mold for immobilizing embryos for stereomicroscope transplants. Dechorionated embryos are dropped individually into wells made by casting this mold into agarose in a 90mm Petri dish. The dimensions of the wells are shown.
Figure 2:Mounting embryos for gastrula-stage transplants. A: ideal shape of a gastrula transplant pipette. A bevel with a sharp tip aids in penetrating the embryo. B: Immobilizing donor and host embryos in methylcellulose. A strip of methylcellulose is laid down in the well of a depression slide and flooded with a generous amount of embryo medium. Embryos are added to the embryo medium and are rolled onto the methylcellulose using a small loop (C). D-G enlargement of embryos in Fig. 1B. D: The donor embryo is oriented to allow easiest access for the pipette, since cells at this stage are still uncommitted. E-G: Shield-stage host embryos are oriented so that the target region is uppermost. Cells transplanted into the boxed regions will contribute to dorsal hindbrain and cranial neural crest derived from the left (E) and right (G) sides or to the ventral hindbrain and spinal cord (F).
Figure 3: Representative images of chimeric embryos.
(A-C) 18hpf chimeric embryos generated by transplantation at shield stage shown in dorsal view, anterior to the left. (A,B) Sorting functions for cell surface receptor EphA4 revealed by analysis of chimeric embryos. Left panels: merge of rhodamine-labeled (rhod.) donor cells (red) and transgenic GFP expressed in specific hindbrain segments (green). (A) WT cells contribute to the entire hindbrain of a control chimeric embryo. (B) EphA4-depleted donor cells are excluded from specific segments in the hindbrain of a WT host. (C) Donor cells targeted to different parts of the neural tube in shield-stage transplants. Donor cells (red) targeted to the forebrain/midbrain (left panel) or hindbrain/spinal cord (right panel) of a WT host counter-stained with an EfnB2a antibody that marks the forebrain, mid-hindbrain boundary and the middle segment of the hindbrain (green). Scale bars: 50μm.
The ease with which transplantation can be used to produce targeted chimeras is one of the great powers of the zebrafish as a vertebrate model. A modification of this protocol not described above allows analysis of maternal gene function for genes with essential roles in zygotic development. Since these mutant fish cannot survive until adulthood, it is necessary to transfer the mutant germline into an otherwise wild-type host embryo, creating “germline clones” of mutant cells. Generating germline mosaics involves transplanting primordial germ cells from a mutant donor to a wild-type host embryo. Unlike all other cell lineages in the embryo, the germ cell lineage is specified very early in development by the inheritance of maternal determinants 5,6 . Thus, the first challenge of making germline mosaics is identifying the primordial germ cells (PGCs) in the donor embryo. At midblastula stages the PGCs reside along the margin 7,8 , so picking up 50-100 random cells from the margin of a lineage-labeled donor embryo frequently results in the transfer of PGCs. When transferred to the animal pole of an unlabeled host embryo, the primordial germ cells actively migrate towards the presumptive gonad where they can be unambiguously identified at 24 hours of development by their characteristic position, large size, and dye retention due to their slow proliferative rate 9-11 . Meanwhile, donor-derived non-PGCs will acquire the fate determined by their location in the host: primarily forebrain and eyes if they were transplanted to the blastula animal pole. Injecting the host embryo with morpholinos that knock down the dead end gene effectively eliminate the host germline in a cell-autonomous manner, so that even a single donor-derived PGC can repopulate the entire germline 10,12 (C.M., personal observation). More recently, a transgenic line that allows PGCs to be visualized in live embryos during blastula stages has been developed 13. When crossed into the mutant whose maternal function is to be determined, this transgene, combined with the dead end morpholinos, will make germline transplants facile because single PGCs can be identified and transplanted into a germline-depleted host.
Thanks to the members of the Moens lab for helpful input during the writing of this article. H.K. is post-doctoral fellow supported by NIH/NICHD grant #5R01HD037909-08. C.B.M. is an investigator with the Howard Hughes Medical Institute.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Injection rig | Applied Scientific Instrumentation | MPPI-3 | Foot pedal can be ordered separately | |
Pronase | Sigma | P5147 | “protease type XIV” | |
Pen-Strep | Sigma | P4458 | 100x stock | |
Methyl cellulose | Sigma | M-0387 | ||
Fluorescent dextrans | Molecular Probes/ Invitrogen | various | Eg. lysine-fixable rhodamine dextran (D7162) | |
Injection pipettes (1.2mmx0.94mmx10cm) | Sutter | BF120-94-10 | ||
Transplant pipette option 1 | World Precision Instruments | TW100-4 | ||
Transplant pipette option 2 | VWR | #53508-400 | ||
micromanipulator | Narishige | UMJ-3FC (?) | ||
Micromanipulator magnetic stand | Narishige | GJ-1 | ||
Transplant apparatus | Sutter Instrument Co. | MI-10010 | ||
Fine micromanipulator | Narishige | MMO-203 | ||
Depression slides | Fisher | 12560A | ||
Agar molds for injection and transplants | AL-AN Mfg, Inc. |
Reagents: Recipes for many of the reagents used for these procedures are found in The Zebrafish Book, which is available in full online at http://zfin.org/zf_info/zfbook/zfbk.html. Recipes are provided there for preparation of Fish Water, Embryo Medium with and without antibiotics, pronase for dechorionating, fluorescent dextrans, and methyl cellulose.